A CA(+) Pair Adjacent to a Sheared GA or AA Pair Stabilizes Size-Symmetric RNA Internal Loops

[Image: see text] RNA internal loops are often important sites for folding and function. Residues in internal loops can have pK(a) values shifted close to neutral pH because of the local structural environment. A series of RNA internal loops were studied at different pH by UV absorbance versus temperature melting experiments and imino proton nuclear magnetic resonance (NMR). A stabilizing CA pair forms at pH 7 in the [Image: see text] and [Image: see text] nearest neighbors when the CA pair is the first noncanonical pair (loop-terminal pair) in 3 × 3 nucleotide and larger size-symmetric internal loops. These [Image: see text] and [Image: see text] nearest neighbors, with CA adjacent to a closing Watson−Crick pair, are further stabilized when the pH is lowered from 7 to 5.5. The results are consistent with a significantly larger fraction (from ∼20% at pH 7 to ∼90% at pH 5.5) of adenines being protonated at the N1 position to form stabilizing wobble CA(+) pairs adjacent to a sheared GA or AA pair. The noncanonical pair adjacent to the GA pair in [Image: see text] can either stabilize or destabilize the loop, consistent with the sequence-dependent thermodynamics of GA pairs. No significant pH-dependent stabilization is found for most of the other nearest neighbor combinations involving CA pairs (e.g., [Image: see text] and [Image: see text]), which is consistent with the formation of various nonwobble pairs observed in different local sequence contexts in crystal and NMR structures. A revised free-energy model, including stabilization by wobble CA(+) pairs, is derived for predicting stabilities of medium-size RNA internal loops.